PROJECT SUMMARY Inducing a robust state of tolerance that is stably maintained despite adverse perturbations is essential for the ability of tolerance mechanisms to maintain the acceptance of the transplanted organ for the life of a recipient. Successfully induced tolerant states in the clinic can be persistent, however in a subset of patients, long-term surviving allografts can be lost. This loss has been correlated with pre-transplant donor-specific antibodies, and in some cases, occurred after episodes of infection. These observations underscore the need to identify individuals who have achieved robust tolerance from those who achieved a less robust/metastable tolerance, where infections may trigger alloreactivity and allograft rejection, and where interventions or closer monitoring may be necessary. While many factors are likely to contribute to a fully robust state of tolerance, in the current funding period, we focused our investigations on the mechanisms that enforce donor-reactive T cell tolerance induced to fully mismatched heart allografts with anti-CD154 in combination with donor spleen cells in mice. We determined that multiple redundant induced T cell tolerance mechanisms maintained transplant tolerance in this model, namely constrained donor-reactive T cell numbers and T cell receptor avidities to levels approximating those in nave mice; induced cell-intrinsic hypo-responsiveness and expression of negative co-inhibitory molecules by the persisting donor-specific T conventional cells; and increased percentages of FoxP3+ cells within the donor-specific CD4+ T cells. We also demonstrated that pro- inflammatory cytokines produced during infection overrode some of the T cell tolerance mechanisms, resulting in allograft rejection, but that tolerance returned spontaneously when the infection was cleared. Finally, we showed that the restored tolerance after infection was eroded in quality and more susceptible to reversal. That established tolerance mechanisms remain responsive to environmental cues highlights the need for tolerance to be robust at initiation as well as resilient, in order for it to successfully maintain graft acceptance for the life of the recipient. Recipient sensitization and the ensuing immunological memory is currently considered the most important barrier to achieving tolerance in the clinic. Clinical studies have identified previous transplant rejection and pregnancy as important sensitizing events. This proposal builds on our current findings and focuses on how prior sensitization after graft rejection (Aim 1) or pregnancy (Aim 2) prevents the establishment of specific T cell tolerance mechanisms. The insights gained from these studies will provide granularity into how T cell tolerance mechanisms cooperate to mediate robust transplant tolerance, guide the identification of biomarkers for robust tolerance, and ultimately, lead to rationally designed therapies that are tailored to achieving robust tolerance in sensitized recipients or for restoring robust tolerance after erosion by infection.